Human tonsil implants xenotransplanted in SCID mice display broad lymphocytic diversity and cellular activation profile similar to those in the original lymphoid organ

BACKGROUND: Models consisting of human immune cells in suspension transferred to severe combined immune deficient (SCID) mice have been invaluable for studying immune response, autoimmunity, and lymphomagenesis. The dissemination of human cells within the mouse body hampers immune functionality with time and favorites the development of human graft vs. mouse host (GvH) disease. To circumvent these limitations we surgically implanted tonsil pieces subcutaneously in SCID animals (hu-ton-SCID mice). Recall humoral responses was elicited and animals did not suffer from signs of GvH disease. A detailed cell subset and cell activation analysis of implants has not yet been reported. METHODS: Implants from 86 hu-ton-SCID mice were evaluated by immunohistochemistry and flow cytometry analyses to assess human lymphoid cell subpopulation surviving with time after implantation, and to evaluate status of human cell activation. Results: B cells persist over 3 months in implants. The proportion of class and type-specific Ig+ cells varied between implants, but as a whole IgG+ cells were more abundant than IgA+, and IgM+ cells, and kappa+ cells predominated over lambda+ cells. The mean proportions of these cells resemble those in the original tonsil. Fine analysis of CD19+ B cells demonstrated no expansion of activated (CD5+, CD23+, CD69+) B cells in implants compared with tonsils, and a decrease of CD19+CD77+ B cells corresponding to a centroblastic phenotype, which is consistent with the disappearance of follicular structure in implants. Double positive CD20+CD27+ memory B cells were detected in implants by immunohistochemistry. T cell CD4+CD8-/CD4-CD8+ ratios were about 4 in implants, that is similar to those in tonsils, and there was no expansion of CD3+CD4+CD8+ and of CD3+CD4-CD8- T-cell subpopulations. T cells activation markers (CD25, CD69) were similarly expressed in implants and tonsils, and implants contained cells with a memory T cell phenotype (CD45RO). Finally cells within implants depicted a low rate of proliferation when assessed by Ki-67 expression levels. Conclusions: Compared with original tonsils, tonsil implants in hu-ton-SCID mice lose the germinal center architecture, which is correlated with the decrease of CD77+ B cells, but conserve T and B cell subpopulation diversity, notably memory cells. In addition, implant T and B cells are not differently activated when compared with those in original tonsils and do not proliferate extensively. These observations indicate indirectly absence of GvH reaction at the cellular level in this model. Collectively, the detailed implant cellular characterization in the hu-ton-SCID model provides a strong rationale for the use of this model in the study of human recall antibody response.     

Porcine complement regulators protect aortic smooth muscle cells poorly against human complement-induced lysis and proliferation: consequences for xenotransplantation

BACKGROUND: Accelerated atherosclerosis after transplantation has been observed and is characterized by smooth muscle cell proliferation in the graft. Porcine cells are frequently used in models of atherosclerosis and porcine organs are considered for use in transplantation. Complement (C) activation is known to play a major role in rejection of xenografts and is also considered to play a role in the development of atherosclerosis. The aim of this study was to investigate the expression and function of membrane bound regulators of complement (CReg) on porcine aortic smooth muscle cells (PASMC). METHODS: The PASMC were assessed for expression of CReg and susceptibility to lysis by human C by flow-cytometry. The effect of various cytokines on CReg expression and C-susceptibility was investigated. The ability of human C to induce cell proliferation was assessed using the Alamar blue assay. RESULTS: The PASMC only express the CReg membrane cofactor protein (MCP) and CD59 on their cell surface. MCP expression was increased by interleukin (IL)-4. In contrast to porcine aortic endothelial cells (PAEC), PASMC were found to be surprisingly sensitive to C-mediated lysis, mainly due to a low level of expression of CD59. Human C-induced proliferation of PASMC, which was dependent on complete membrane attack complex (MAC) formation. CONCLUSIONS: Endogenously expressed CReg on PASMC poorly protect these cells to human C. Human C can induce proliferation of PASMC. In order to prevent accelerated atherosclerosis in porcine xenografts, increased levels of CReg not only have to be obtained on the endothelial cells but also on the smooth muscle cells.     

From ABO-incompatible human kidney transplantation to xenotransplantation

The development (in 1981) of a protocol for successful renal allotransplantation across ABO barriers is outlined. From this experience, the concept of "adaptation", subsequently termed "accommodation", was defined. It was then hypothesized that a similar approach might allow pig-to-human organ xenotransplantation. This hypothesis was explored in the pig-to-baboon renal transplantation model, with graft survival for a maximum of 23 days. Rejection episodes were temporarily reversed, providing encouragement that discordant xenotransplantation would one day prove successful. Finally, the preparation of the thymokidney, developed as a means of inducing xenotolerance, is briefly reviewed.     

Blockade of indirect recognition mediated by CD4+ T cells leads to prolonged cardiac xenograft survival

Abstract:  The T-cell response to xenografts is induced by direct and indirect recognition of xenoantigens. Although the importance of indirect recognition is well established in vitro, the contribution of this pathway to xenograft rejection in vivo remains to be fully elucidated. We herein investigated the direct contribution of indirect recognition to cardiac xenograft rejection in the rat-to-mouse (PVG.R8-to-C57BL/10) concordant model. Rat xenoantigens invoked a vigorous proliferative response in mouse T cells harvested from naïve or graft recipients at rejection. Indirect recognition predominated the response, as antibodies against mouse class II I-A^b, CD80, or CD86 molecules significantly (45 to 60%) blocked the proliferative response. Importantly, the blockade of indirect recognition in vivo by treating the graft recipients with a monoclonal antibody (mAb) against class II I-A^b molecule on days 0, 1, and 3 post-transplantation resulted in significant ( P  < 0.009) prolongation of cardiac xenograft survival (Mean Survival Time (MST) >94 ± 55 days vs. 7 ± 0.8 days for controls). In contrast, treatment of recipients with a mAb against mouse class I H-2K^b/D^b molecules did not significantly affect graft rejection (MST = 8 ± 1 days). These results demonstrate that indirect recognition mediated by CD4⁺ T cells plays a critical role in the rejection of cardiac grafts in the rat-to-mouse xenogeneic model.     

Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B isolectin to alphagalactosyl carbohydrate antigens in the surface phase

The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Galalpha1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme-linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Galalpha1-3Gal1-4GlcNAcbeta1-3Galbeta1-4Glc) than for the Galalpha-monosaccharide. The binding properties of MOA and GS I-B(4) to the xenogenic disaccharide (Galalpha1-3Galbeta1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B(4) to the same epitope. Non-xenogenic disaccharide-coupled neoglycoproteins having galactose end groups linked alpha1-2 or alpha1-4 to Gal or linked alpha1-3 to GalNAc bound very weakly to MOA, whereas GS I-B(4) recognized all of these disaccharides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B(4) for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasaccharide and is far more specific in its binding preferences than the Griffonia lectin.     

Understanding of and attitudes to xenotransplantation: a survey among Italian university students

Xenotransplantation is currently at the experimental stages on animal models and many problems still have to be overcome in the biomedical, immunological and ethical fields. Moreover, people's attitudes to xenotransplantation vary: surveys among intensive-care staff have revealed negative opinions, while the general public and students seem to be more positive. Little is known about the influence of schooling and the choice of university faculty on attitudes to xenotransplantation. The aims of this study were: (i) to evaluate university students' attitudes to xenotransplantation; (ii) to investigate any socio-demographic, religious and educational determinants behind students' opinions on xenotransplantation. University undergraduates on five different courses were surveyed at Padua University. A 24-item questionnaire was distributed to students at the end of lectures and completed anonymously immediately after its distribution. No information was given to students beforehand. Statistical analysis: chi-squared, Pearson's test; P-values <0.05 were considered significant. A total of 585 of 602 (97.2%) students completed the questionnaire (132 males, 453 females, mean age 20.4, range 19 to 43 yr). They were on courses in Medicine (33.85%), Agriculture (5.98%), Veterinary Medicine (11.45%), Psychology (18.46%) and Educational Sciences (30.26%). As for their previous schooling, they came from classical or scientific high school (58.3%), technical college (14.7%), language college (6.3%), teacher training college (11.9%) or others (8.8%). Concerning their religious beliefs, 83% were Catholics, and 56.2% defined themselves as practising Catholics. Eighty-eight percentage of the students knew of the possibility of animal organs being transplanted into humans and 77.9% of them approved of this idea. When grouped according to gender and education, a higher proportion of students approving of xenotransplantation were male (P = 0.017) and had attended classical or scientific high school (P = 0.011). Disapproval for moral, ethical or religious reasons was higher among practising than among non-practising Catholics; the latter rejected xenotransplantation more for immunological and infectious reasons (P = 0.014). As for the type of university course, a higher proportion of students approving of xenotransplantation attended science courses (Veterinary Medicine, Agriculture and Medicine vs. Educational Sciences and Psychology) (P = 0.013). University students generally approved of xenotransplantation. Male gender and a high-school education were associated with a greater acceptance of xenotransplantation. Practising vs. non-practising Catholics reported significantly different reasons for any disapproval of xenotransplantation. The choice of a science rather than an arts faculty at university was more strongly associated with a positive opinion on xenotransplantation.     

The graft content of hyaluronan is increased during xenograft rejection

BACKGROUND: Hyaluronan, a macromolecule with strong water binding capacity, is associated with interstitial oedema during rejection of allogeneic transplants. However, the involvement of hyaluronan during xenograft rejection has previously not been investigated. The aims of this study were to characterize hyaluronan content and distribution during rejection of concordant mouse-to-rat cardiac xenografts, and to explore the effects of hyaluronidase (HAse) on xenograft survival. METHODS: Graft recipients were treated with 15-deoxyspergualin (DSG) or both HAse and DSG. Grafts were removed on day 5 from some of the animals to analyse hyaluronan and water content, while other animals were used to investigate graft survival. The hyaluronan content was measured by a radiometric assay and the distribution was analysed by histochemical staining. RESULTS: In xenografts undergoing rejection (the DSG group) there was a strong increase of the hyaluronan [555 +/- 93 microg/g dry weight (dw)] and water (82.7 +/- 0.4%) contents compared with normal mouse heart tissue (166 +/- 10 microg/g dw; P < 0.01 and 78.6 +/- 0.5%; P < 0.001, respectively). The combined use of HAse and DSG reduced the accumulation of hyaluronan (284 +/- 43 microg/g dw; P < 0.05 vs. DSG) but did not affect the average water content. The average graft survival time did not differ between the groups; however, three grafts in the HAse + DSG-treatment group survived much longer than the longest-surviving grafts in the DSG group. CONCLUSIONS: These data suggest that the graft content of hyaluronan considerably increases during xenograft rejection. HAse effectively reduces this accumulation, but does not affect the average water content.